Oligonucleotide arrays for gene expression are increasingly becoming popular. The ability to produce mass arrays either by spotting pre-synthesized oligonucleotides or by in situ synthesis such as photolithographic means reliably and to perform gene expression studies reproducibly using such arrays has generated great deal of enthusiasm in the microarray research community. Increasingly, gene expression profiling is becoming a mainstream tool for molecular diagnosis of genomic disorders such as cancer. Oligonucleotide arrays also overcome some practical difficulties encountered with cumbersome process of generating CDNA arrays such as a prerequisite for mRNA source, massive parallel RT-PCR reactions, cloning and sequence verifications.
There are several different labeling protocols that researchers employ in studying gene expression on the cDNA arrays, but when applied to oligo arrays they do not work to satisfaction. Listed below are some common methods researchers use to generate probes in a microarray gene expression experiment. 1) Labeling during first strand cDNA synthesis using either wild type or modified reverse transcriptase (either using oligo dT primers and/or random primers). 2) “Modified Eberwine method”, a strategy that relies on generating RNA probes by labeling during RNA synthesis and has the advantage of RNA amplification. The strategy is cumbersome and requires multiple steps that require the conversion of the RNA into DNA with a suitable promoter for enabling amplification. Further the process requires at least 4 different enzymes, ranging from RT, RNA polymerase, DNA ligase, RNase H and other additives. Additionally, the exponential nature of amplification has the potential to skew the experimental results. 3) Labeling, post first strand synthesis by chemical coupling methods to allyl amino modified nucleotides is another accepted method. The advantages of these labeling methods for cDNA arrays is that a large proportion of the probes made are generally complementary to the extensive region (typically about 500 or more bases) on the arrays and therefore produce acceptable signal for studies.
However, the probes generated by the above methods will require a fragmentation step prior to use in oligo array experiments owing to limitations from hybridization thermodynamics of larger fragments of nucleic acid on solid support. Not only is the process laborious and cumbersome, fragmentation also renders a large proportion of labeled synthesized probes useless, because they lack the corresponding complementary region on the arrayed oligos. Hence, signal generated from such fragmented probes are likely to be a few orders of magnitude diminished, consequently resulting in difficulties with detection.